A selected lipid type virus, viral hepatitis, has been recognized as an important and serious sequela of parenteral exposure to blood and blood components since the early 1940s. It was originally believed that all such blood-associated hepatitis was caused by the serum hepatitis virus (now called the Hepatitis B virus, or HBV). Subsequently, the development of sensitive assays for infection with this virus revealed that only approximately one-third of transfusion-associated hepatitis was caused by the HBV. It was thought that the remaining hepatitis was caused by the Hepatitis A virus (HAV). However, the development of sensitive assays for HAV led to the recognition of a new hepatitis virus, the non-A, non-B hepatitis virus (NANB) in 1975. The successful application of sensitive screening tests for HBV to blood donors has resulted in a decrease (but not disappearance) of HBV in transfusion-associated hepatitis; at present approximately 90% of such hepatitis is caused by non-A, non-B agents.
Similarly, hepatitis following administration of plasma protein derivatives such as antihemophilic factor was thought to be caused solely by HBV. However, in the late 1970s, the association of NANB agents with administration of antihemophilic factor to hemophiliacs was reported and confirmed. As with transfusion-associated hepatitis, the application of serologic screening methods to plasma donors has resulted in a relative decrease in the importance of HBV in such blood product-associated hepatitis.
Non-A, non-B hepatitis is the major cause of transfusion associated hepatitis in the United States Presently, less than 10% of post-transfusion cases are caused by the hepatitis B virus. Of the remainder, cytomegalovirus may account for a small proportion but the vast majority are caused by an as yet unidentified agent. There is a large amount of evidence supporting a transmissible agent as the cause of NANBH. This includes transmission studies done in both humans and non-human primates. Chimpanzees and marmoset monkeys have both been shown to be susceptible to infection by at least some NANBH agents. Though very costly and cumbersome to work with, these animals can be used to aid in the characterization of the infectious agent of NANBH.
Unfortunately, serologic tests for the detection of NANB agents are not available for detection of potentially infectious donors because the agents have not been adequately identified and characterized despite extensive efforts to do so. Therefore, blood and plasma protein derivative products remain potential sources for transmission of hepatitis agents to recipients. The resultant hepatitis can be quite serious, even life-threatening, and can result in not only acute hepatitis but also chronic hepatitis in a significant proportion of cases.
For these reasons, attempts to inactivate hepatitis agents in blood and plasma products have been pursued with vigor. Such approaches have included the use of heat, the addition of anti-HBV antibody, the use of solid immunoadsorbents or other chemical-specific adsorbents, exposure to ultraviolet radiation, the addition of certain inactivating substances, such as beta-propriolactone, surface-active substances, etc. None of the approaches has been entirely successful and some have introduced an added potential risk (e.g., beta-propriolactone is carcinogenic). Failure of these approaches stems from relative resistance of the agents to physical or chemical inactivation, particularly when in the presence of high protein concentrations as occurs with blood products and from limited knowledge about the nature of the hepatitis agents, especially the NANB agents.
As part of a systematic characterization of NANB agents by standardized virologic methods, the present inventors first established that HBV and at least one NANB agent contain lipids essential for the integrity and viability of the viruses. This was established by exposing the viruses to a potent lipid solvent (chloroform) and demonstrating that such chloroform-extracted viruses were rendered non-infectious in a suitable susceptible host, the chimpanzee (Pan troglodytes).
It was later found that when viruses were contained in a dried protein carrier such as a commercial factor VIII concentrate preparation, the viruses were much more difficult to inactivate with the lipid solvent than when the protein carrier was reconstituted to a liquid with water and then treated with the lipid solvent. It was also found that the biologic activity of the protein carrier was difficult to maintain when it was treated as a liquid with the lipid solvent. Thus, this invention expands the parent invention by modifying the process in such a way that a lipid virus contained a dry protein carrier, in particular a plasma derivative, can be readily and consistently inactivated by a lipid solvent while the biologic activity of the plasma product is maintained at a high level.
The present invention relates to a method of inactivating lipid viruses that frequently contaminate a plasma protein product. Said viruses are most frequently hepatitis B virus and non-A, non-B hepatitis virus but are also defined to include members of the herpesvirus group (cytomegalovirus, Epstein-Barr virus, herpes zoster virus, herpesvirus type 1 and type 2), the delta agent (a type of non-A, non-B hepatitis virus), togaviruses (including rubella virus), bunyaviruses, retroviruses (including the human T-cell leukemia viruses), orthomyxoviruses (including influenza), paramyxoviruses (measles, mumps), rhabdoviruses (rabies, Marburg agent), arenaviruses (Lassa fever, other hemorrhagic fevers), coronaviruses, hepadnaviruses, and poxvirus group (smallpox, vaccinia virus). Other viruses, known or suspected, such as the putative agent of acquired imune deficiency syndrome (AIDS) are included as viruses possibly containing essential lipids. (Evidence that the agent of AIDS is a retrovirus has recently been published.)
The plasma protein product is defined as a protein derived from blood or blood plasma that is intended for human medical uses most often to correct a deficiency of that particular blood protein, or as an aid to treating some disease that might benefit from an increased concentration of the particular blood protein. Over 100 such plasma proteins have been identified and perhaps many more will eventually be found (cf. Putnam, Plasma Proteins, pp. 36-41 and Table 1). Examples of such blood products are antihemophilic factor (factor VIII), factor IX, fibrinogen, fibronectin, albumin, complement components, plasminogen, transferrin, and haptoglobin, and many other plasma proteins that have indicated medical uses but that may not at this time be marketed. In general, lipoproteins would not survive the process of this invention in their native state. In addition, certain blood proteins that are not intended for therapeutic use but may be used, for example, in diagnostic tests, may also be treated to reduce their hazard to the person handling them. Such plasma protein products are often dried by lyophilization during the manufacture process in order to preserve their biologic potency, increase shelf life and for ease of handling and shipping. The residual moisture content of such dried products ranges between about 0.5 and 1.5%.
In the present invention the contaminating lipid containing viruses are inactivated by treating (extracting or contacting) the dry plasma product with a lipid solvent in which water has been dissolved such that the lipid solvent contains between about 75% to 100% of the amount of water required to reach its dissolved water saturation point. The preferred lipid solvent is chloroform (CHCl.sub.3) or CHCl.sub.3 and a lower alcohol (e.g., methanol or ethanol), or the fluorocarbons (trichlorotrifluoroethane) which include the most common agents such as CCl.sub.3 F, CH.sub.2 F.sub.2, CCl.sub.2 F.sub.2, CCl.sub.2 FCClF.sub.2 and others sold under the trademark registration Freon.RTM. or Genetron.RTM.. Throughout the present invention the biologic activity of the plasma protein is retained but the infectivity of the virus is removed.
The period of time for the treatment is about 10 minutes to about 10 hours and at least about 10 minutes. The temperature of the treatment is from about 4.degree. C. to 40.degree. C.
The quantity of lipid solvent used to treat dried plasma product is from about equal to the weight of the dry blood product to about 1,000 times the weight of the dry blood product.
The lipid solvent or the treating agent may be removed from the plasma product by evaportion with a stream of pure, dry nitrogen gas (N.sub.2), by vacuum evaporation or by a combination of these or other physical methods which returns the plasma product to the dry state and free of the lipid solvent.
Commercial chloroform contains 0.5% (v/v) ethanol as a stabilizer. The quantity of dissolved water required to saturate pure chloroform varies between 0.019% (wt/v) at 3.degree. C. to 0.065% at 22.degree. C. to 0.118% at 43.degree. C. (Stephen and Stephen (eds.), Solubilities of Inorganic and Organic Compounds, Vol. 1, MacMillan Co., New York, 1963, p. 370). Therefore, the absolute quantity of water in the chloroform can be increased by increasing the temperature. In addition, the quantity of dissolved water can be increased by increasing the ethanol concentration.
It has been found that dry (water-free) chloroform is inconsistently effective in inactivating vaccinia virus. It is believed that increases in the dissolved water content of the treating agent tend to increase the effectiveness of the inactivation treatment, with superior results achieved as the water dissolved in the treating agent approaches 100% saturation of the treating agent at the temperature of treatment. It is believed that inactivation treatments should be conducted at saturation levels of from about 75 to about 100%. A two-phase system including water and treating agent, while relatively effective in inactivating virus, may destroy activity of blood products, such as Factor VIII.